Vaccine preparation and immunization
Fusion-GEM complexes (10 µg per mouse) were mixed with a complex adjuvant of ISA 201 VG (Seppic, Paris, France) and poly(I:C) (Sigma, St. Louis, MN, USA) based on our previous research [15]. Each milliliter of vaccine contained 350 µl of fusion-GEM complexes, 100 µl of 2 mg/ml poly(I:C) and 550 µl of ISA 201 VG. Female BALB/c mice (6-8 weeks) were randomly divided into 4 groups (n=18/group): 3 vaccine groups and 1 control group. Vaccine groups received the vaccines (ZI-△-PA-GEM, ZI-JE-△-PA-GEM or ZI-GP-△-PA) intramuscularly (IM). Control group received PBS mixed with adjuvant. Nine mice from each group were boosted twice at 2-week intervals. Serum was collected at 0, 4, 6 and 8 weeks post-immunization (wpi) and heat inactivated at 56°C for 30 min.